Acute limb ischemia reperfusion injury (LIRI) is characterized by an imbalance in the free radicals nitric oxide and superoxide anion. Accordingly, replenishment of nitric oxide and removal of superoxide are each partially effective, but greater protection of muscle injury is expected from the simultaneous correction of both abnormalities. To address this unmet clinical need, Radikal Therapeutics (RTX) is developing R-100, a novel small molecule agent formed from the covalent linkage of an organic nitrovasodilator that releases nitric oxide, and a pyrrolidine nitroxide that acts as a superoxide dismutase mimetic, catalase mimic, and peroxynitrite decomposition catalyst. In combination, these functionalities allow R-100 to remove toxic reactive oxygen species and deliver nitric oxide without the confounding effect of producing peroxynitrite. In a Phase 1 SBIR, we demonstrated in a severe murine model of acute LIRI that R-100 resuscitation profoundly reduces limb histologic damage, lipid peroxidation, neutrophil infiltration, and % of injured muscle. Given the centrality of these pathophysiologic mechanisms to clinical LIRI, our Phase 1 SBIR data justify progression to the Phase 2 SBIR scope of work.
Aim #1 : Rank order the potency of R-100 and its analogues in a rodent model of LIRI R- 100 (both enantiomers) will be compared to 10 analogues in a murine LIRI screening model. The top 3 performing agents will be compared over their full dose-responses (20, 40, 80 mg/kg/day) in order to annoint the best performer as the candidate molecule (CM).
Aim #2 : Establish the pharmacodynamic (PD) profile of the CM in a porcine model of acute LIRI. We will scale-up and manufacture >99% pure CM in to support large animal efficacy studies and follow-on GLP toxicology and safety pharmacology investigations. In a large animal model of acute LIRI (n=6 minipigs per group) induced by an endovascular balloon occlusion of the femoral artery for 6 h, we will establish the dose-response of parenteral CM (0, 10, 30, 80 mg/kg/day TID) initiated 10 min before reperfusion via direct arterial infusion, f0llowed by an IV infusion thereafter. At 7, 14, and 21 days after reperfusion we will correlate limb perfusion (by Doppler flow), nerve conduction velocity (""""""""NCV"""""""", per electromyography, """"""""EMG""""""""), and muscle strength (by foot pressure on a walking surface) with simultaneous plasma concentrations of CM in order to construct a PD profile.
Aim #3 : Establish the acute safety, toxicity, and tolerance of IV CM in the minimum set of GLP toxicology and safety pharmacology studies required for FDA IND application. We will carry out the minimum set of required FDA-mandated IND-enabling GLP studies to support application for a first-in-man, dose-escalation, single-dose investigation of IV CM in healthy human volunteers. Genetic toxicology studies will include: the Ames, chromosomal aberration, and rat micronucleus assays. Safety pharmacology investigations will include: 1) rat neurobehavioral and respiratory studies, and 2) canine cardiovascular studies. IV dose range-finding and 2 week repeat-dose toxicology studies in rats and dogs, with associated toxicokinetic determinations.
Loss of blood flow to a limb is a medical emergency requiring immediate restoration of perfusion. Therapies that reopen blocked vessels may paradoxically induce further tissue damage, known as reperfusion injury. There are no approved therapies to prevent or treat this condition. We are developing a novel drug that targets the basic mechanisms of reperfusion injury and is intended as a prophylactic agent in patients undergoing revascularization to restore blood flow to the limb. We will test this agent in a clinically-relevat large animal model of acute limb ischemia, to establish its utility in this setting, and carry out series of toxicology and safety pharmacology studies to define its toxicity, in preparation for an FDA-approved Phase 1 clinical trial in human beings.
